Soil alkalinity greatly affects plant growth and crop productivity. Although RNA-Seq analyses have been conducted to investigate genome-wide gene expression in response to alkaline stress in many plants, the expressions of alkali-responsive genes in rice have not been previously investigated. In this study, the transcriptomic data between an alkaline-tolerant (WD20342) and an alkaline-sensitive (Caidao) rice cultivar were compared under alkaline stress conditions. A total of 962 important alkali-responsive (IAR) genes from highly differentially expressed genes (DEGs) were identified, including 28 alkaline-resistant cultivar-related genes, 771 alkaline-sensitive cultivar-related genes and 163 cultivar-non-specific genes. Gene ontology (GO) analysis indicated the enrichment of IAR genes involved in various stimulus or stress responses. According to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the IAR genes were related primarily to plant hormone signal transduction and biosynthesis of secondary metabolites. Additionally, among these 962 IAR genes, 74 were transcription factors and 15 occurred with differential alternative splicing between the different samples after alkaline treatment. Our results provide a valuable resource on alkali-responsive genes and should benefit the improvement of alkaline stress tolerance in rice.